Characterization of the nuclear export signal of polypyrimidine tract-binding protein.

The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, we have shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for its activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor.     

Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes

The translation of the unspliced and partially spliced viral mRNAs that encode the late, structural proteins of HIV-1 depends on the viral-protein Rev. Oligomeric binding of Rev to the Rev response element (RRE) in these mRNAs promotes their export from the nucleus and thus controls their expression. Here, we compared the effects of hydrophobic to hydrophilic mutations within the oligomerization domain of Rev using assays for oligomeric RNA binding, protein structure, and export from the nucleus. Oligomeric RNA binding alone does not correlate well with RNA transport activity in the subset of mutants. However, protein structure as judged by CD spectroscopy does correlate well with Rev function. The oligomeric assembly of Rev-L18T is impaired but exhibits minor defects in structure and retains a basal level of activity in vivo. The prevalence of L18T in infected individuals suggests a positive selection mechanism for L18T modulation of Rev activity that may delay the onset of AIDS.     

REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.     

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP

Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated, ending instead in a conserved stem-loop sequence. Histone pre-mRNAs lack introns and are processed in the nucleus by a single cleavage step, which produces the mature 3' end of the mRNA. We have systematically examined the requirements for the nuclear export of a mouse histone mRNA using the Xenopus oocyte system. Histone mRNAs were efficiently exported when injected as mature mRNAs, demonstrating that the process of 3' end cleavage is not required for export factor binding. Export also does not depend on the stem-loop binding protein (SLBP) since mutations of the stem-loop that prevent SLBP binding and competition with a stem-loop RNA did not affect export. Only the length of the region upstream of the stem-loop, but not its sequence, was important for efficient export. Histone mRNA export was blocked by competition with constitutive transport element (CTE) RNA, indicating that the mRNA export receptor TAP is involved in histone mRNA export. Consistent with this observation, depletion of TAP from Drosophila cells by RNAi resulted in the restriction of mature histone mRNAs to the nucleus.     

Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632

We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5' splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle.     

Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export‐competent RNP

The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap‐dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E‐sensitivity element (4E‐SE), and its overexpression alters the nuclear export of several eIF4E‐sensitive mRNAs. LRPPRC‐mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E‐binding site and it coincides with the subcellular re‐distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E‐ and eIF4E‐sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.     

Polypyrimidine tract-binding proteins are cleaved by caspase-3 during apoptosis

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.     

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.     

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.     

The human insulin receptor mRNA contains a functional internal ribosome entry segment

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.     

Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.